METHOD FOR CULTURING PRIMARY CELLS OF GASTRIC CANCER AND GALLBLADDER AND BILE DUCT CANCER, AND SUPPORTING REAGENTS

Abstract

A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. The core of the technology is that: (1) the solid tumor tissues of gastric cancer and gallbladder cancer and cholangiocarcinoma are treated with a mild cell dissociation reagent, and the primary tumor cells of gallbladder cancer and cholangiocarcinoma in a bile sample are isolated by a mild method to ensure the vitality of cancer cells to the greatest extent; (2) a special serum-free medium is prepared, and tumor cells of gastric cancer and gallbladder cancer and cholangiocarcinoma are cultured in vitro by a suspension culture system to eliminate the interference of normal cells to the greatest extent while ensuring normal amplification of cancer cells.

Claims

1. A primary cell culture medium for gastric cancer and/or gallbladder cancer and cholangiocarcinoma, is composed of antibiotic-antimycotic, HEPES, GlutaMax, non-essential amino acids, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein FGF-10, human recombinant protein Wnt-3a, human recombinant protein Noggin, SB202190, A83-01, Primocin, N-acetyl-L-cysteine, Nicotinamide, N-2 Supplement, cortisol, B27, ITS-X, Gastrin 1, Y-27632 and Advanced DMEM/F12 culture medium; wherein, the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL; the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL; the final concentration of amphotericin B in the antibiotic-antimycotic is 250-250 ng/mL; the final concentration of the HEPES is 8-12 mM; the final concentration of the GlutaMax is 0.8-1.2% (volume percentage); the concentration of glycine in the non-essential amino acids is 80-120 μM; the concentration of L-alanine in the non-essential amino acids is 80-120 μM; the concentration of L-asparagine in the non-essential amino acids is 80-120 μM; the concentration of L-aspartic acid in the non-essential amino acids is 80-120 μM; the concentration of L-glutamic acid in the non-essential amino acids is 80-120 μM; the concentration of L-proline in the non-essential amino acids is 80-120 μM; the concentration of L-serine in the non-essential amino acids is 80-120 μM; the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein FGF-10 is 5-25 ng/mL; the final concentration of the human recombinant protein Wnt-3a is 200-300 ng/mL; the final concentration of the human recombinant protein Noggin is 100-200 ng/mL; the final concentration of the SB202190 is 5-10 μM; the final concentration of the A83-01 is 0.25-1.25 μM; the final concentration of Primocin is 1% (volume percentage); the final concentration of the N-acetyl-L-cysteine is 0.5-2 mM; the final concentration of the Nicotinamide is 5-10 mM; the final concentration of the N-2 Supplement is 1% (volume percentage); the final concentration of the cortisol is 20-50 ng/mL; the final concentration of the B27 is 1.5-2.5% (volume percentage); the final concentration of the ITS-X is 0.8-1.2% (volume percentage); the final concentration of the Gastrin 1 is 8-12 nM; the final concentration of the Y-27632 is 5-20 μM; and the rest is the Advanced DMEM/F12 medium.

2. A kit of reagents for culturing primary cells of gastric cancer and/or gallbladder cancer and cholangiocarcinoma, which is any of the following: (A1) Consisting of the culture medium and all or part of the following: sample dissociation solution, sample preservation solution and sample washing solution; The sample dissociation solution is composed of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I is 150-250 U/mL; the final concentration of the collagenase II is 150-250 U/mL; the final concentration of the collagenase IV is 50-150 U/mL; and the rest is PBS; The sample preservation solution is composed of fetal bovine serum, antibiotic-antimycotic, HEPES and HBSS; wherein the final concentration of the fetal bovine serum is 1-5% (volume percentage); the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL; the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μm/mL; the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL; the final concentration of the HEPES is 8-12 mM; and the rest is HBSS; The sample washing solution is composed of antibiotic-antimycotic and PBS; wherein the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL; the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL; the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL; and the rest is PBS; (A2) Consisting of the culture medium and the cell isolation buffer; The cell isolation buffer is composed of P/S, heparin sodium and PBS; wherein the final concentration of penicillin in the P/S is 100-200 U/mL; the final concentration of streptomycin in the P/S is 100-200 μg/mL; the final concentration of the heparin sodium is 10 IU/mL; and the rest is PBS; (A3) Consisting of (A1) and all or part of the following reagents: cell digestion solution, digestion termination solution and cell cryopreserving solution; (A4) Consisting of (A2) and all or part of the following reagents: cell digestion solution, digestion termination solution and cell cryopreserving solution; The composition of the cell digestion solution is as follows: each 10 mL of the cell digestion solution contains 4-6 mL of Accutase, EDTA with a final concentration of 5 mM and 1.5-2.5 mL of TrypLE Express, and the rest is PBS; The digestion termination solution is composed of fetal bovine serum, antibiotic-antimycotic and DMEM culture medium; wherein the final concentration of the fetal bovine serum is 8-12% (volume percentage); the final concentration of penicillin in the antibiotic-antimycotic is 100-200 U/mL; the final concentration of streptomycin in the antibiotic-antimycotic is 100-200 μg/mL; the final concentration of amphotericin B in the antibiotic-antimycotic is 250-500 ng/mL; and the rest is the DMEM culture medium; The cell cryopreserving solution is composed of Advanced DMEM/F12 medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 medium, the DMSO and the 1% methylcellulose solution is 20:2: (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose with a concentration of 1 g/100 ml.

3. (canceled)

4. A method for culturing primary cells of gastric cancer and/or gallbladder cancer and cholangiocarcinoma is either method A or method B: Method A: A method for culturing primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma, comprising the following steps: (a1) dissociating solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with the sample dissociation solution, to obtain primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma; (a2) suspension-culturing the dissociated primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma in step (a1) with the medium; Method B: A method for culturing primary tumor cells in a bile sample of gallbladder cancer and cholangiocarcinoma, comprising the following steps: (b1) separating primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma, to obtain primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma; (b2) suspension-culturing the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma separated in step (b1) with the medium.

5. The method according to claim 4, characterized in that: in step (a1), the solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma are dissociated with the sample dissociation solution according to the method comprising the following steps: according to the dosage of 0.1-0.3 mL of sample dissociation solution per mg of tissue, treating the trimmed solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with the sample dissociation solution preheated at 37° C., dissociating the sample at 37° C. for 15 minutes to 3 hours; In step (b1), the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma are separated from the bile sample of gallbladder cancer and cholangiocarcinoma according to a method comprising the following steps: suspending the cells in the bile sample of gallbladder cancer and cholangiocarcinoma with the cell isolation buffer, and then obtaining the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma by density gradient centrifugation.

6. The method according to claim 4, characterized in that: in step (a2), the primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma are suspension-cultured with the medium according to a method comprising the following steps: suspension-culturing the primary cells in solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with the medium using a cell culture vessel M under the condition of 37° C., 5% CO.sub.2, and replacing the medium every 2-4 days; In step (b2), the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma are suspension-cultured with the medium according to a method comprising the following steps: suspension-culturing the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma with the medium using a cell culture vessel M under the condition of 37° C., 5% CO.sub.2, and replacing the medium every 2-4 days; The cell culture vessel M is any of the following: (I) cell culture vessels made of polystyrene, cell culture vessels made of polycarbonate, cell culture vessels made of polymethylmethacrylate, cell culture vessels made of COC resin, cell culture vessels made of cycloolefin polymer or cell culture vessels with a low attachment surface; (II) cell culture vessels after CYTOP modification on the cell culture vessels in (I).

7. The method according to claim 6, characterized in that: In (II), the cell culture vessels in (I) are CYTOP-modified according to a method comprising the following steps: performing pure oxygen etching on the cell culture vessels in (I) at an etching power of 20 W for 3 minutes; then covering the surface of the cell culture vessels with 1% CYTOP solution, and drying the 1% CYTOP solution in the air to complete CYTOP modification; The composition of the 1% CYTOP solution is as follows: each 100 mL of the 1% CYTOP solution contains 1 mL of CYTOP, and the rest is fluorocarbon oil.

8. The method according to claim 4, characterized in that: before step (a1), the following steps of predissociation treatment the solid tumor tissues of gastric cancer and/or gallbladder cancer and cholangiocarcinoma are also included: washing the surface of a solid tumor tissue sample of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with 70-75% ethanol (volume percentage); washing the solid tumor tissue sample of gastric cancer and/or gallbladder cancer and cholangiocarcinoma successively with the sample washing solution and the sterile PBS solution

9. The method according to claim 8, characterized in that: the in vitro isolation time of the solid tumor tissue sample of gastric cancer and/or gallbladder cancer and cholangiocarcinoma for the predissociation treatment is within 2 hours, and the solid tumor tissue sample of gastric cancer and/or gallbladder cancer and cholangiocarcinoma is preserved in the sample preservation solution before the predissociation treatment.

10. The method according to claim 4, characterized in that: in step (a1), the following steps are also included after dissociation treatment of the solid tumor tissue of gastric cancer and/or gallbladder cancer and cholangiocarcinoma with the sample dissociation solution: terminating the dissociation reaction with the digestion termination solution, and collecting the cell suspension; filtering the cell suspension and removing tissue fragments and adherent cells; centrifuging and then resuspending cells with sterile PBS; centrifuging again, then resuspending the cell precipitation with the medium.

11. The method according to claim 4, characterized in that: before step (a2), the following step are also included: passaging the primary cells in solid tumor of gastric cancer and/or gallbladder cancer and cholangiocarcinoma when masses with a diameter of 50-80 μm are formed by the primary cells in solid tumor of gastric cancer and/or gallbladder cancer and cholangiocarcinoma; In step (b2), the following steps are also included: passaging the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma when masses with a diameter of 50-80 μm are formed by the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma.

12. The method according to claim 11, characterized in that: the cell digestion solution used for the passage is the cell digestion solution.

13. The method according to claim 11, characterized in that: the digestion termination solution used for the passage is the digestion termination solution.

14. The method according to claim 4, characterized in that: the method also comprise steps of cryopreserving and/or resuscitating the primary cells in solid tumor of gastric cancer and/or gallbladder cancer and cholangiocarcinoma or the primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma after 2-3 passages and amplifications; The cell cryopreserving solution for the cryopreserving is the cell cryopreserving solution.

15-17. (canceled)

18. The culture medium according to claim 1, characterized in that: the gastric cancer is primary gastric cancer; and the gallbladder cancer and cholangiocarcinoma is primary gallbladder cancer and cholangiocarcinoma.

19. The culture medium according to claim 1, characterized in that: the gastric cancer is a metastatic lesion of gastric cancer; and the gallbladder cancer and cholangiocarcinoma is a metastatic lesion of gallbladder cancer and cholangiocarcinoma.

20. The culture medium according to claim 1, characterized in that: the primary cells of gastric cancer are primary cells in solid tumor of gastric cancer; and the primary cells of gallbladder cancer and cholangiocarcinom are primary cells in solid tumor of gallbladder cancer and cholangiocarcinoma or primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma.

21. The culture medium according to claim 1, characterized in that: the primary cells of gastric cancer are isolated from surgical samples of patients with gastric cancer; and the primary cells of gallbladder cancer and cholangiocarcinoma are isolated from a surgical sample, a puncture sample or a bile sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0118] FIG. 1 illustrates the single cells obtained from a gastric cancer tissue after treatment. The scale is 100 μm, subject to 100× magnification.

[0119] FIG. 2 illustrates the cell masses obtained from a gastric cancer tissue after primary culture. The scale is 100 μm, subject to 100× magnification.

[0120] FIG. 3 illustrates a HE staining image of a gastric cancer cell mass section obtained from a gastric cancer tissue after primary culture. The scale is 100 μm, subject to 200× magnification.

[0121] FIG. 4 illustrates an immunofluorescence staining image of cancer cell masses obtained from a gastric cancer tissue after primary culture. The scale is 50 μm, subject to 200× magnification.

[0122] FIG. 5 illustrates a copy number variation (CNV) analysis based on the sequencing results, showing that the copy number variation of all generations of primary cell cultures of gastric cancer (P1, P2, P3, P4 and P5) is highly consistent with that of the primary tumor tissue (Tumor) of gastric cancer.

[0123] FIG. 6 illustrates the results of an in vitro drug sensitivity test on the primary cells of gastric cancer cultured by using the present invention.

[0124] FIG. 7 illustrates the single cells obtained from a cholangiocarcinoma tissue after treatment. The scale is 100 μm, subject to 100× magnification.

[0125] FIG. 8 illustrates the cell masses obtained from a cholangiocarcinoma tissue after primary culture. The scale is 100 μm, subject to 100× magnification.

[0126] FIG. 9 illustrates a HE staining image of a cholangiocarcinoma cell mass section obtained from a cholangiocarcinoma tissue after primary culture. The scale is 100 μm, subject to 200× magnification.

[0127] FIG. 10 illustrates an immunohistochemical staining image of a cancer cell mass paraffin section obtained from a cholangiocarcinoma tissue after primary culture. The scale is 100 μm, subject to 200× magnification.

[0128] FIG. 11 illustrates the single cells obtained from a cholangiocarcinoma bile sample after treatment. The scale is 100 μm, subject to 100× magnification.

[0129] FIG. 12 illustrates the cell masses obtained from a cholangiocarcinoma bile sample after primary culture. The scale is 100 μm, subject to 100× magnification.

[0130] FIG. 13 illustrates a HE staining image of a cholangiocarcinoma cell mass section obtained from a cholangiocarcinoma bile sample after primary culture. The scale is 100 μm, subject to 100× magnification.

[0131] FIG. 14 illustrates an immunohistochemical staining image of a cancer cell mass paraffin section obtained from a cholangiocarcinoma bile sample after primary culture. The scale is 50 μm, subject to 100× magnification.

[0132] FIG. 15 illustrates a design view of a microplate chip of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0133] The following embodiments are intended for a better understanding of the present invention, but not limiting the present invention. Unless otherwise specified, all the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, all the test materials used in the following embodiments are available from the conventional biochemical stores. All the quantitative tests in the following embodiments are provided with three repeated experiments, and the average value of the results is taken.

Embodiment 1. Preparing a Reagent for Culturing Primary Cells of Gastric Cancer

[0134] 1. Sample Preservation Solution (100 mL)

[0135] The specific formula of sample preservation solution (100 mL) is as shown in Table 1.

TABLE-US-00001 TABLE 1 Sample preservation solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Fetal bovine Gibco#16000-044 2 mL 2% serum Antibiotic- Gibco#15240062 1 mL 1% antimycotic HEPES Gibco# 15630080 1 mL 10 mM HBSS Gibco# 14170161 Replenish to 100 mL

[0136] After being prepared, the sample preservation solution was divided in 15 mL centrifuge tubes, and each tube was filled with 5 mL. After aliquoting, the sample preservation solution can be preserved at 4° C. for 1 month.

[0137] 2. Sample Washing Solution (100 mL)

[0138] The specific formula of sample washing solution (100 mL) is as shown in Table 2.

TABLE-US-00002 TABLE 2 Sample washing solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Antibiotic- Gibco#15240062 1 mL 1% antimycotic PBS Gibco#21-040-CVR Replenish to 100 mL

[0139] The sample washing solution is to be prepared just before use.

[0140] 3. Sample Dissociation Solution (10 mL)

[0141] The specific formula of sample dissociation solution (10 mL) is as shown in Table 3.

TABLE-US-00003 TABLE 3 Sample dissociation solution (10 mL) Final Reagent Brand Article No. Dosage Concentration 10× collagenase I 10× stock solution 1 mL 200 U/mL 10× collagenase II 10× stock solution 1 mL 200 U/mL 20× collagenase IV 20× stock solution 0.5 mL 100 U/mL PBS Gibco#21-040-CVR Replenish to 10 mL

[0142] Note: The sample dissociation solution is to be prepared just before use.

[0143] In Table 3, the preparation of collagenase stock solution is as shown in Tables 4-6.

TABLE-US-00004 TABLE 4 10×collagenase I stock solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Collagenase I Gibco +19017100-017 1 g 2000 U/mL PBS Gibco#21-040-CVR Replenish to 100 mL

[0144] After being prepared, the 10×collagenase I stock solution was divided in 1.5 mL sterile centrifuge tubes, and each tube was filled with 1 mL. The stock solution can be kept at −20° C. for a long term.

TABLE-US-00005 TABLE 5 10×collagenase II stock solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Collagenase II Gibco # 17101-015 1 g 2000 U/mL PBS Gibco#21-040-CVR Replenish to 100 mL

[0145] After being prepared, the 10×collagenase II stock solution was divided in 1.5 mL sterile centrifuge tubes, and each tube was filled with 1 mL. The stock solution can be kept at −20° C. for a long term.

TABLE-US-00006 TABLE 6 20×collagenase IV stock solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Collagenase IV Gibco #17104-019 1 g 2000 U/mL PBS Gibco#21-040-CVR Replenish to 100 mL

[0146] After being prepared, the 20×collagenase IV stock solution was divided in 1.5 mL sterile centrifuge tubes, and each tube was filled with 1 mL. The stock solution can be preserved at −20° C. for a long term.

[0147] In Tables 4, 5 and 6, the unit U of the collagenase (the collagenase I or the collagenase IV) is defined by the enzyme activity of protease: 1 μmol of L-leucine can be released by treating the collagenase (the collagenase I or the collagenase IV) with 1 U of protease for 5 hours at 37° C. and pH 7.5.

[0148] 4. Cell Digestion Solution (10 mL)

[0149] The specific formula of cell digestion solution (10 mL) is as shown in Table 7.

TABLE-US-00007 TABLE 7 Cell digestion solution (10 mL) Final Reagent Brand Article No. Dosage Concentration Accutase Gibco#A11105-01 5 mL 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM TrypLE Express Gibco#12604013 2 mL PBS Gibco#21-040-CVR Replenish to 10 mL

[0150] The cell digestion solution is to be prepared just before use.

[0151] 5. Digestion Termination Solution (100 mL)

[0152] The specific formula of digestion termination solution (100 mL) is as shown in Table 8.

TABLE-US-00008 TABLE 8 Digestion termination solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Fetal bovine serum Gibco#16000-044 10 mL 10% Antibiotic-antimycotic Gibco#15240062 1 mL  1% DMEM culture Gibco#11965-092 Replenish medium to 100 mL

[0153] The prepared digestion termination solution can be preserved at 4° C. for 1 month.

[0154] 6. Primary Cell Culture Medium for Solid Tumor Tissues of Gastric Cancer (100 mL)

[0155] The specific formula of primary cell culture medium for solid tumor tissues of gastric cancer (100 mL) is as shown in Table 9.

TABLE-US-00009 TABLE 9 Primary cell culture medium for solid tumor tissues of gastric cancer (100 mL) Final Reagent Brand Article No. Dosage Concentration Antibiotic- Gibco#15240062 1 mL 1% antimycotic HEPES Gibco#15630080 1 mL 10 mM GlutaMax Gibco#35050061 1 mL 1% Non-essential Gibco#11140050 1 mL 1% amino acids 1000× human 1000× stock 50-500 μL 10-100 ng/mL recombinant solution protein EGF 1000× human 1000× stock 50-250 μL 10-50 ng/mL recombinant solution protein bFGF 1000× human 1000× stock 25-125 μL 5-25 ng/mL recombinant solution protein HGF 1000× human 1000× stock 25-125 μL 5-25 ng/mL recombinant solution protein FGF-10 1000× human 1000× stock 100-150 μL 200-300 ng/mL recombinant solution protein Wnt-3a 1000× human 1000× stock 100-200 μL 100-200 ng/mL recombinant solution protein Noggin SB202190 1000× stock 50-100 μL 5-10 μM solution A83-01 100000× 1-5 μL 0.25-1.25 μM stock solution Primocin ™ Invivogene#ant- 1 mL 1% pm-1 N-acetyl-L- 1000× stock 100-400 μL 0.5-2 mM cysteine solution Nicotinamide 1000× stock 100-200 μL 5-10 mM solution N-2 Supplement Gibco#17502001 1 mL 1% Cortisol 1000× stock 80-200 μL 20-50 ng/mL solution B27 Gibco#12587010 2 mL 2% ITS-X Gibco#51500056 1 mL 1% Gastrin 1000× stock 100 μL 10 nM solution Y-27632 1000× stock 50-200 μL 5-20 μM solution Advanced Gibco#12634010 Replenish to DMEM/F12 100 mL culture medium

[0156] After being prepared, the primary cell culture medium for solid tumor tissues of gastric cancer was filtered and sterilized with a 0.22 μM syringe-driven filter (Millipore SLGP033RS). The culture medium can be preserved at 4° C. for two weeks.

[0157] In Table 9, the preparation of human recombinant protein stock solution is as shown in Table 11-Table 16, the preparation of SB202190 stock solution is as shown in Table 17, the preparation of A83-01 stock solution is as shown in Table 18, the preparation of N-acetyl-L-cysteine stock solution is as shown in Table 19, the preparation of Nicotinamide stock solution is as shown in Table 20, the preparation of cortisol stock solution is as shown in Table 21, the preparation of gastrin stock solution is as shown in Table 22, and the preparation of Y-27632 stock solution is as shown in Table 23. The preparation of 100× BSA solution required in preparation of these stock solutions is as shown in Table 10.

TABLE-US-00010 TABLE 10 100× BSA solution (1 mL) Final Reagent Brand Article No. Dosage Concentration BSA Sigma#A1933 0.1 g 0.1 g/mL PBS Gibco#21-040-CVR Replenish to 1 mL

[0158] 100×BSA solution is to be prepared just before use.

TABLE-US-00011 TABLE 11 1,000× human recombinant protein EGF stock solution (5 mL) Final Reagent Brand Article No. Dosage Concentration Human recombinant Peprotech 100 μg 20 μg/mL protein EGF AF-100-15-100 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Replenish to 5 mL

[0159] After being prepared, the 1,000× human recombinant protein EGF stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00012 TABLE 12 1,000× human recombinant protein bFGF stock solution (2.5 mL) Final Reagent Brand Article No. Dosage Concentration Human recombinant Peprotech  50 μg 20 μg/mL protein bFGF AF-100-18B-50 100× BSA solution 250 μL 0.01 g/mL PBS Gibco#21-040-CVR Replenish to 2.5 mL

[0160] After being prepared, the 1,000× human recombinant protein bFGF stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00013 TABLE 13 1,000× human recombinant protein HGF stock solution (5 mL) Final Reagent Brand Article No. Dosage Concentration Human recombinant Peprotech 100 μg 20 μg/mL protein HGF AF-100-39-100 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Replenish to 5 mL

[0161] After being prepared, the 1,000× human recombinant protein HGF stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00014 TABLE 14 1,000× human recombinant protein FGF-10 stock solution (5 mL) Final Reagent Brand Article No. Dosage Concentration Human recombinant Peprotech 100 μg 20 μg/mL protein FGF-10 AF-100-26-100 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Replenish to 5 mL

[0162] After being prepared, the 1,000× human recombinant protein FGF-10 stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00015 TABLE 15 1,000× human recombinant protein Wnt-3a stock solution (2.5 mL) Final Reagent Brand Article No. Dosage Concentration Human R&D 5036-WN-500 500 μg 200 μg/mL recombinant protein Wnt-3a 100× BSA 250 μL 0.01 g/mL solution PBS Gibco#21-040-CVR Replenish to 2.5 mL

[0163] After being prepared, the 1,000× human recombinant protein Wnt-3a stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00016 TABLE 16 1,000× human recombinant protein Noggin stock solution (5 mL) Final Reagent Brand Article No. Dosage Concentration Human recombinant Shanghai nearshore 500 μg 100 μg/mL protein #C018 Noggin 100× BSA solution 500 μL 0.01 g/mL PBS Gibco#21-040-CVR Replenish to 5 mL

[0164] After being prepared, the 1,000× human recombinant protein Noggin stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −80° C. for a long term.

TABLE-US-00017 TABLE 17 1,000× SB202190 stock solution (1.51 mL) Final Reagent Brand Article No. Dosage Concentration SB202190 Sigma#S7067 5 mg 10 mM DMSO Replenish to 1.51 mL

[0165] After being prepared, the 1,000×SB202190 stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00018 TABLE 18 100,000× A83-01 stock solution (1.05 mL) Final Reagent Brand Article No. Dosage Concentration A83-01 Tocris#2939 10 mg 25 mM DMSO Replenish to 1.05 mL

[0166] After being prepared, the 1,000×A83-01 stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00019 TABLE 19 1,000× N-acetyl-L-cysteine stock solution (5 mL) Final Reagent Brand Article No. Dosage Concentration N-acetyl-L-cysteine Sigma#A9165 0.41 g 0.5 M Ultrapure water Replenish to 5 mL

[0167] After being prepared, the 1,000×N-acetyl-L-cysteine stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00020 TABLE 20 1,000× Nicotinamide stock solution (4 mL) Final Reagent Brand Article No. Dosage Concentration Nicotinamide Sigma#N0636 2.44 g 5 M Ultrapure water Replenish to 4 mL

[0168] After being prepared, the 1,000×Nicotinamide stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00021 TABLE 21 1,000× cortisol stock solution (100 mL) Final Reagent Brand Article No. Dosage Concentration Cortisol Sigma#H0888 2.5 mg 25 μg/mL Absolute ethyl Sigma#E7023   5 mL 5% alcohol Ultrapure water Replenish to 100 mL

[0169] After being prepared, the 1,000× cortisol stock solution was divided in 1.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00022 TABLE 22 1,000× gastrin stock solution (48 mL) Final Reagent Brand Article No. Dosage Concentration Gastrin NJPeptide#Pep12307 1 mg 10 μM Ultrapure Replenish to 48 mL water

[0170] After being prepared, the 1,000× gastrin stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

TABLE-US-00023 TABLE 23 1,000× Y-27632 stock solution (3.125 mL) Final Reagent Brand Article No. Dosage Concentration Y-27632 MCE#129830-38-2 10 mg 10 mM Ultrapure Replenish to 3.125 mL water

[0171] After being prepared, the 1000×Y-27632 stock solution was divided in 0.5 mL sterile centrifuge tubes. The stock solution can be preserved at −20° C. for a long term.

[0172] 7. Cell Cryopreserving Solution

[0173] The specific formula of cell cryopreserving solution is as shown in Table 24.

TABLE-US-00024 TABLE 24 Cell cryopreserving solution Reagent Brand Article No. Dosage Advanced DMEM/F12 culture medium Gibco#12634010 20 mL DMSO Sigma#D2438  2 mL 1% methylcellulose solution  1 mL

[0174] The cell cryopreserving solution is to be prepared just before use.

[0175] In Table 24, the preparation of 1% methylcellulose solution is as shown in Table 25.

TABLE-US-00025 TABLE 25 1% methylcellulose solution (10 mL) Final Reagent Brand Article No. Dosage Concentration Methylcellulose Sigma#M7027 0.1 g 10 g/L Ultrapure water Replenish to 10 mL

[0176] The prepared 1% methylcellulose solution can be preserved at 4° C. for a long time.

[0177] 8. 1% CYTOP Solution

TABLE-US-00026 TABLE 26 1% CYTOP solution (100 mL) Final Reagent Brand Article No. Dosage Concentration CYTOP Asashi glass#CTL-809M  1 mL 1% Fluorocarbon oil 3M# FC40 Replenish to 100 mL

[0178] The prepared 1% CYTOP solution can be preserved at a normal temperature for a long time.

Embodiment 2. Obtaining Postoperative Specimen of Gastric Cancer

[0179] 1. The inventor cooperated with national triple A, first-class hospitals in China, and the cooperation passed formal medical ethical review.

[0180] 2. The attending physician selected the enrolled patients according to the clinical indications specified in the medical guidelines, and selected suitable samples for in vitro culture according to the intraoperative clinical indications. The selection criteria of samples were as follows: primary gastric cancer, with pathological staging of stage II, III or IV, gastric cancer or metastatic lesions of various pathological types, and samples with the surgical specimen of gastric cancer weighing more than 20 mg.

[0181] 3. The attending physician provided the patient's basic clinical information, such as gender, age, medical history, family history, smoking history, pathological stages and types, and clinical diagnosis. The information related to patient privacy, such as patient's name and ID number, was concealed and replaced by a unified experimental number, and the naming principle of the experimental number was the 8-digit date of sample collection+last four digits of the patient's admission number. For example, for a sample provided on Jan. 1, 2018, when the patient's admission number was T001512765, then the sample experiment number was 201801012765.

[0182] 4. During the operation, a surgeon collected a fresh specimen in a sterile environment of an operating room and placed it in a sample preservation solution prepared in advance (see Embodiment 1). After being detached, the sample was temporarily stored on ice and transported to a laboratory within two hours for the next operation.

Embodiment 3. Performing Predissociation Treatment for Tissue Sample of Gastric Cancer

[0183] The following steps were operated on ice, and the whole operation process was completed within 10 minutes.

[0184] All the surgical apparatuses used in the following actions were used only after high-temperature and high-pressure sterilization and oven drying in advance.

[0185] 1. The sample was weighed.

[0186] 2. The sample surface was washed with 75% (volume percentage) ethanol for 10 to 30 seconds.

[0187] 3. The sample was washed with sample washing solution for 10 times and with sterile PBS solution for 5 times.

[0188] 4. Fatty tissues, connective tissues and necrotic tissues in the sample were carefully peeled off with ophthalmic scissors, ophthalmic forceps, scalpel and other apparatuses.

Embodiment 4. Dissociating Tissue Sample of Gastric Cancer

[0189] All the surgical apparatuses used in the following actions were used only after high-temperature and high-pressure sterilization and oven drying in advance.

[0190] 1. The tissues were cut into small pieces of about 1 mm.sup.3 with ophthalmic scissors.

[0191] 2. The tissue sample cut into pieces was treated with sample dissociation solution preheated to 37° C. in advance according to the dosage of 0.1 mL of sample dissociation solution (see Embodiment 1) per mg of tissue, and the sample was dissociated at 37° C. for 15 minutes to 3 hours. The dissociation of the sample was observed under a microscope every 15 minutes until a large number of individual cells were observed.

[0192] 3. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 1), and the cell suspension was collected.

[0193] 4. The cell suspension was filtered with a 40 μm sterile cell strainer to remove tissue fragments and adherent cells.

[0194] 5. The cell suspension was centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0195] 6. The cells were resuspended with 5 mL of sterile PBS and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0196] 7. The cell precipitation was resuspended with the primary cell culture medium for solid tumor tissues of gastric cancer (see Embodiment 1), the cell state was observed under a microscope, and the cells were counted.

[0197] In addition to tumor cells, there were also a large number of various types of other cells, such as red blood cells, lymphocytes and fiber cells, mixed in the single cell suspension obtained by dissociation, as shown in FIG. 1. One of the advantages of the present method is that only the cancer cells could be amplified in the subsequent culture process, the proportion of other cells gradually decreases or even disappears, and primary tumor cells of gastric cancer with a high purity are finally obtained.

Embodiment 5. Culturing Primary Cells of Gastric Cancer

[0198] 1. A low-attachment-surface was used for suspension-culturing primary cells of gastric cancer, and the culture medium in use was the primary cell culture medium for solid tumor tissues of gastric cancer in Embodiment 1 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein noggin was 100 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM); a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

[0199] 2. The cell state was observed every day, and the culture medium was replaced every 3 days until the cells formed masses with a diameter of about 80 μm.

[0200] As shown in FIG. 2, after 3-10 days of culture, cancer cells were amplified, forming cell masses with a diameter of 80 μm, the total number of tumor cells exceeded 10.sup.7, and the number of other types of cells was significantly reduced or even disappeared. After a large number of sample tests, the success rate of the present method for culturing in vitro primary tumor cells of gastric cancer could reach 80%.

Embodiment 6. Passaging Primary Cells of Gastric Cancer

[0201] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0202] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0203] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 1) and digested at 37° C. The digestion of cell masses was observed under the microscope every 5 minutes until all the cell masses were digested into individual cells.

[0204] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 1), and the cell suspension was collected.

[0205] 5. The cell suspension was centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0206] 6. The cell precipitation was resuspended with the primary cell culture medium for solid tumor tissues of gastric cancer, and the cells were counted.

[0207] 7. A low-attachment-surface was used for culturing primary cells of gastric cancer, and the culture medium in use was the primary cell culture medium for solid tumor tissues of gastric cancer in Embodiment 1; a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 7. Cryopreserving Primary Cells of Gastric Cancer

[0208] The primary cells of gastric cancer suspension-cultured could be frozen after 2-3 passages and amplifications:

[0209] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0210] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0211] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 1) and digested at 37° C. The digestion of cell masses was observed under the microscope every 15 minutes until all the cell masses were digested into individual cells.

[0212] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 1), the cell suspension was collected, and the cells were counted.

[0213] 5. The cell suspension was centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0214] 6. The cell precipitation was resuspended at a density of 10.sup.6/mL with cell cryopreserving solution (see Embodiment 1), each 2 mL cryopreserving tube contained 1 mL of cell suspension, and the cell suspension was frozen overnight in a gradient cooling box and transferred into liquid nitrogen for long-term preservation.

Embodiment 8. Resuscitating Primary Cells of Gastric Cancer

[0215] Primary cells of gastric cancer preserved in liquid nitrogen were resuscitated:

[0216] 1. 37° C. sterile water was prepared five minutes in advance.

[0217] 2. The cryopreserving tubes were removed from liquid nitrogen, and the cells were quickly melted in 37° C. sterile water.

[0218] 3. The cell solution was centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0219] 4. The cell precipitation was resuspended with the primary cell culture medium for solid tumor tissues of gastric cancer (see Embodiment 1), the primary cells of gastric cancer were cultured using a low-attachment-surface, the cells in each tube were resuscitated into a 3.5 cm culture dish, and the cells were cultured in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 9. HE Staining Identification of Primary Cells of Gastric Cancer

[0220] Description of reagent consumables to be used in the following embodiment:

[0221] HE Staining Kit (Beijing Solarbio Science & Technology Co., Ltd., #G1120);

[0222] Cationic anticreep slide (ZSGB-BIO);

[0223] Xylene, methanol, acetone (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure);

[0224] Neutral resin adhesive (Beijing Yili Fine Chemicals Co., Ltd.).

[0225] 1. Primary cell masses from solid tumor tissues of gastric cancer obtained by culturing with the primary cell culture medium for solid tumor tissues of gastric cancer in Embodiment 1 (wherein the final concentration of human recombinant protein EGF was 20 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of human recombinant protein Noggin was 100 ng/mL; the final concentration of SB202190 was 5 μM; the final concentration of A83-01 was 1 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were collected by centrifugation at 800 g, and immobilized with 4% paraformaldehyde. Cell mass precipitation was embedded in paraffin and sectioned with a thickness of 5 μm.

[0226] 2. Paraffin sections were immersed in dimethylbenzene solution and incubated at a room temperature for 5 minutes for dewaxing, and after this process was repeated for 3 times, the sections were flushed with deionized water twice.

[0227] 3. The sections were immersed in absolute ethanol and incubated at a room temperature for 10 minutes, and this process was repeated twice.

[0228] 4. The sections were immersed in 95% ethanol and incubated at a room temperature for 10 minutes, and after this process was repeated twice, the sections were flushed with deionized water twice.

[0229] 5. 100 μL of hematoxylin staining solution was added for staining for 1 min when the moisture on the slide was slightly dried.

[0230] 6. The hematoxylin staining solution was absorbed, and the slide was washed for 3 times with tap water.

[0231] 7. 100 μL of differentiation solution was added dropwise for differentiation for 1 min.

[0232] 8. The differentiation solution was absorbed, the slide was washed twice with tap water and washed with distilled water once.

[0233] 9. The moisture on the slide surface was absorbed, and 200 μL of eosin stain was added dropwise for staining for 40 s.

[0234] 10. The eosin stain was absorbed, and rinsing and dehydration were performed successively with 75%, 80%, 90% and 100% ethanol for 20 s, 20 s, 40 s and 40 s.

[0235] 11. After the ethanol was dried in the air, 50 μL of dimethylbenzene was added dropwise for cell permeability.

[0236] 12. After dimethylbenzene was completely dried, a drop of neutral resin adhesive was added, and the slide was sealed with a cover glass, observed under the microscope and photographed.

[0237] FIG. 3 shows an HE staining effect image of primary tumor cells of gastric cancer obtained by in vitro culture. It can be seen that these cells generally have the characteristics of cancer cells, such as high nuclear cytoplasmic ratio, hyperchromasia, chromatin condensation in nucleus, multinucleation and uneven cell size, and dozens to hundreds of tumor cells gather to form tumor cell masses with a certain three-dimensional structure.

Embodiment 10. Immunofluorescence Staining Identification of Primary Cells of Gastric Cancer

[0238] Description of reagents to be used in the following embodiment:

[0239] Paraformaldehyde (Beijing Beihua Zhongtuo Technology Co., Ltd. analytical pure), paraformaldehyde powder was dissolved with ultrapure water to form 4% (4 g/100 mL paraformaldehyde solution;

[0240] Methanol and dimethyl sulfoxide (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure);

[0241] Hydrogen peroxide (Beijing Beihua Zhongtuo Technology Co., Ltd., 35%);

[0242] Methanol, dimethyl sulfoxide and 35% hydrogen peroxide were mixed in the ratio of 4:4:1 (volume ratio) to form Dan's rinsing solution;

[0243] Bovine serum albumin (Sigma, #A1933), bovine serum albumin was dissolved with PBS solution to form 3% (3 g/100 mL) BSA solution;

[0244] Immunofluorescence primary antibody (Abcam, #ab17139);

[0245] Immunofluorescence secondary antibody (CST, #4408);

[0246] Hoechst staining solution (Beijing Solarbio Science & Technology Co., Ltd., #C0021);

[0247] The cell masses of gastric cancer obtained by culturing with the primary cell culture medium for solid tumor tissues of gastric cancer in Embodiment 1 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 25 ng/mL; the final concentration of human recombinant protein HGF was 25 ng/mL; the final concentration of human recombinant protein FGF-10 was 25 ng/mL; the final concentration of human recombinant protein Wnt-3a was 300 ng/mL; the final concentration of human recombinant protein Noggin was 200 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were subject to immunofluorescence staining according to the following steps, the primary antibody was CK8+CK18, and epithelial-derived cells were characterized.

[0248] 1. The cell masses in the culture dish were collected and washed once with PBS, the cell precipitation was resuspended with 4% paraformaldehyde, and immobilized at 4° C. overnight.

[0249] 2. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cell precipitation was resuspended with precooled methanol solution and placed on ice for 1 hour.

[0250] 3. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cell precipitation was resuspended with Dan's rinsing solution and placed at a room temperature for 2 hours.

[0251] 4. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cells were washed with 75%, 50% and 25% (volume percentage) methanol solution diluted with PBS for 10 minutes each time.

[0252] 5. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cell precipitation was suspended with 3% BSA solution and sealed at a room temperature for 2 hours.

[0253] 6. The primary antibody was diluted with 3% BSA solution in a ratio of 1:500, the cell precipitation was resuspended with antibody diluent (3% BSA solution), and the primary antibody was placed at 4° C. overnight.

[0254] 7. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cell precipitation was washed for 5 times with PBS solution, 20 minutes each time.

[0255] 8. The secondary antibody was diluted with 3% BSA solution in a ratio of 1:2,000, the cell precipitation was resuspended with antibody diluent (3% BSA solution), and the secondary antibody was placed at a room temperature for 2 hours.

[0256] 9. The cell solution was centrifugated at 800 g, the supernatant was discarded, and the cell precipitation was washed for 5 times with PBS solution, 20 minutes each time.

[0257] 10. 100×Hoechst staining solution was added in a volume ratio of 1/100 for staining at a room temperature for 20 minutes.

[0258] 11. The cell precipitation was washed twice with PBS solution, 10 minutes each time. The staining of cell masses was observed using a laser confocal microscope.

[0259] FIG. 4 shows an immunofluorescence staining effect image of primary tumor cell masses of gastric cancer cultured in vitro. It can be seen that all the cells constituting the cell masses are CK8/CK18 positive and epithelial-derived, which confirms that the tumor cells obtained by the present method are those with a high purity. Immunofluorescence staining identification was performed on 20 primary cultures of gastric cancer samples, and the statistical results showed that the proportion of tumor cells in the primary cells of gastric cancer obtained in the present method reached 70%-93% (Table 27).

TABLE-US-00027 TABLE 27 Immunofluorescence staining identification of primary cultures of gastric cancer samples S/N Sample No. Positive Rate of CK8/CK18 1 20180305DHJ0296 74.9% 2 201803055YL9928 73.8% 3 20180306T5R7222 71.5% 4 20180306ZY5276 81.4% 5 20180306ZXY5195 70.5% 6 20180306WDH5845 83.2% 7 20180306MX7450 87.3% 8 20180307YXH1978 93.2% 9 20180307CBP3705 86.0% 10 20180308MDX7573 79.6% 11 20180309ZBQ8443 89.8% 12 20180309RDJ7864 93.0% 13 20180309DYF0245 91.5% 14 20180312CZZ0983 92.2% 15 2018031255L1528 92.5% 16 20180314LMY0097 72.6% 17 20180314WYC8956 76.8% 18 20180315HZY9207 92.3% 19 20180315Z5F3973 75.4% 20 20180315YZM0341 70.4%

Embodiment 11. Primary Cell Cultures and Primary Tumor Tissues of Gastric Cancer

[0260] The DNA extraction process mentioned in the following embodiment was performed with TIANGEN blood/tissue/cell genome extraction kit (DP304).

[0261] The library building process mentioned in the following embodiments was performed with NEB DNA sequencing and library building kit (E7645).

[0262] The high-throughput sequencing mentioned in the following embodiments refers to the Illumina HiSeq X-ten sequencing platform.

[0263] 1. A solid tumor tissue sample of gastric cancer was obtained, 10 mg of solid tumor tissue sample of gastric cancer was firstly taken for DNA extraction, library building and whole-genome high-throughput sequencing (WGS), with a sequencing depth of 300× before in vitro culture operations were performed, and the remaining solid tumor tissue samples were used for culturing in vitro primary cells of gastric cancer.

[0264] 2. Gastric cancer tissues were treated and cultured with the primary cell culture medium for solid tumor tissues of gastric cancer in Embodiment 1 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein Noggin was 100 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 1 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 8 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 8 μM) for a period of time to form cell masses with a diameter of more than 100 μm, which were recorded as P0-generation cells, followed by P1, P2, . . . , Pn successively according to the number of passages. 10.sup.6 cells were taken respectively from P1, P2, P3, P4 and P5-generation primary tumor cell cultures of gastric cancer for DNA extraction, library building and whole-genome high-throughput sequencing (WGS), with a sequencing depth of 300×.

[0265] 3. Copy number variation (CNV) analysis was performed on the sequencing results of each group, and the copy number variations between tumor tissues of primary gastric cancer and all generations of primary cell cultures of gastric cancer were compared. As shown in FIG. 5, the copy number variation of all generations of primary cell cultures of gastric cancer (P1, P2, P3, P4 and P5) was highly consistent with that of the tumor tissue (Tumor) of primary gastric cancer, so the primary cells of gastric cancer obtained by the present method could represent the real condition of the patient's primary tumor.

Embodiment 12. Comparing Success Rates of Different Primary Cell Culture Media for Culturing Primary Cells of Gastric Cancer

[0266] The operation methods and processes for culturing primary cells in all samples in this embodiment were identical (as mentioned above), and the only difference was the culture medium formula. Various tested primary cell culture media are shown in Table 28. Wherein Scheme D is the formula used in the present invention, and the details are shown in Table 9 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein Noggin was 100 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 1 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 8 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 8 μM).

TABLE-US-00028 TABLE 28 Primary cell culture medium formula for test (100 mL) Final Culture Brand Concen- Medium Reagent Article No. Dosage tration Scheme mTeSR ™ 1 Stemcell#05850 100 mL A medium Scheme Alveolar Epithelial ScienCell#3201 100 mL B Cell Medium P/S Gibco#15140122  1 mL 1% HEPES Gibco#15630080  1 mL 10 mM 1000× human 1000× stock 250 μL 50 ng/mL recombinant solution protein Scheme EGF C 1000× human 1000× stock 100 μL 20 ng/mL recombinant solution protein bFGF DMEM/F12 Gibco#14170161 Replenish culture medium to 100 mL Scheme See Table 9 D

[0267] After being prepared, the primary cell culture media were filtered and sterilized with a 0.22 μM syringe-driven filter (Millipore SLGP033RS). The primary cell culture media can be preserved at 4° C. for two weeks.

[0268] 20 samples were treated in each of the four primary cell culture medium schemes, the sample treatment and culture operations were performed according to the methods described in Embodiments 3, 4 and 5, and the success rates of those primary cell culture media for culturing primary cells in solid tumor tissues of gastric cancer were counted after 10 days of culture, as shown in Table 29:

TABLE-US-00029 TABLE 29 Culture in different media Primary cell culture Success rate medium scheme Scheme A  0% (0/20) Scheme B  0% (0/20) Scheme C 10% (2/20) Scheme D 75% (15/20)

[0269] It can be seen that different primary cell culture media have an extremely great impact on the success rate for culturing primary cells of gastric cancer, the primary cell culture medium for solid tumor tissues of gastric cancer used in the present invention (Table 9) can stimulate the proliferation of cancer cells in the solid tumor tissue sample of gastric cancer to the greatest extent and improve the success rate for culturing primary cells in solid tumor tissues of gastric cancer.

Embodiment 13. Comparing Success Rates of Different Sample Preservation Solutions for Culturing Primary Cells of Gastric Cancer

[0270] The operation methods and processes for culturing primary cells in all samples in this embodiment were identical (as mentioned above), and the only difference was the sample preservation solution formula. Various tested sample preservation solutions are shown in Table 30. Wherein Scheme E is the formula used in the present invention, and the details are shown in Table 1.

TABLE-US-00030 TABLE 30 Sample preservation solution formula for test (100 mL) Sample preservation Final solution scheme Reagent Brand Article No. Dosage Concentration Scheme A HBSS Gibco#14170161 100 mL P/S Gibco#15140122  1 mL 1% Scheme B HBSS Gibco#14170161 Replenis hto 100 mL Scheme C P/S Gibco#15140122  1 mL 1% DMEM culture Gibco#11965-092 Replenish to medium 100 mL Antibiotic-antimycotic Gibco#15240062  1 mL 1% Scheme D Fetal bovine serum Gibco#16000-044  10 mL 10%  DMEM culture Gibco#11965-092 Replenish to medium 100 mL Scheme E See Table 1

[0271] After being prepared, those sample preservation solutions in the above table were divided in 15 mL centrifuge tubes, and each tube was filled with 5 mL of sample preservation solutions. The sample preservation solution divided can be preserved at 4° C. for 1 month.

[0272] 20 samples were treated in each of the five sample preservation solution schemes, the samples were detached and temporarily preserved at 4° C. in the sample preservation solution; the sample treatment and culture operations were performed according to the methods described in Embodiments 3, 4 and 5 two hours after detachment, and the success rates of those sample preservation solutions for culturing primary cells in solid tumor tissues of gastric cancer were counted after 10 days of culture, as shown in Table 31:

TABLE-US-00031 TABLE 31 Culture in different sample preservation solutions Sample preservation Culture success Bacteria/Fungus solution scheme rate contamination rate Scheme A 45% (9/20) 40% (15/20) Scheme B 55% (11/20) 10% (2/20) Scheme C 60% (12/20) 10% (2/20) Scheme D 70% (14/20)  0% (0/20) Scheme E 75% (15/20)  0% (0/20)

[0273] It can be seen that different sample preservation solutions have a great impact on the culture success rate for culturing primary cells in solid tumor tissues of gastric cancer, the sample preservation solution used in the present invention (Table 1) can protect the activity of cancer cells in the solid tumor tissue sample of gastric cancer to the greatest extent and improve the success rate.

Embodiment 14. Comparing Success Rates of Different Sample Dissociation Solutions for Culturing Primary Cells of Gastric Cancer

[0274] The operation methods and processes of primary culture of all samples in this embodiment were identical (as mentioned above), and the only difference was the sample dissociation solution formula. Various tested sample dissociation solutions are shown in Table 32. Wherein Scheme D is the formula used in the present invention, and the details are shown in Table 3.

TABLE-US-00032 TABLE 32 Sample dissociation solution formula for test (10 mL) Sample dissociation Final solution Reagent Brand Article No. Dosage Concentration Scheme A Trypsin Gibco# 25200056 10 mL Scheme B 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM DNAse Thermo# EN0521 62.5 μL 62.5 U/mL TrypLE ™ Gibco#12604013 Replenish to Express 10 mL Scheme C 20× collagenase IV 20× stock solution 1 mL 200 U/mL DNAse Thermo# EN0521 62.5 μL 62.5 U/mL PBS Gibco#21-040-CVR Replenish to 10 mL Scheme D See Table 3

[0275] The sample dissociation solution is to be prepared just before use.

[0276] 20 samples of solid tumor tissue masses of gastric cancer weighing more than 100 mg were selected and divided into four parts equally, and sample treatment and culture operations were performed with the above four sample dissociation solutions respectively according to the methods described in Embodiments 3, 4 and 5. The success rates of sample dissociation solutions for culturing primary cells in solid tumor tissues of gastric cancer were counted after 10 days of culture, as shown in Table 33 below:

TABLE-US-00033 TABLE 33 Culture in different sample dissociation solutions Scheme Scheme Scheme Scheme S/N Sample No. A B C D 1 20180316WDB9885 Failed Failed Failed Failed 2 20180316WSX0073 Failed Successful Successful Successful 3 20180319P5L9161 Failed Successful Successful Successful 4 20180320YC8122 Failed Failed Failed Successful 5 20180322XJH2568 Failed Failed Successful Successful 6 20180323LAH6704 Failed Failed Failed Failed 7 20180323WYL1663 Failed Failed Failed Successful 8 20180327ZY3082 Failed Failed Successful Successful 9 20180327WB51138 Failed Failed Failed Successful 10 20189327YXH5008 Failed Successful Successful Successful 11 20180327W5Q2382 Failed Failed Failed Failed 12 20180328WFM4371 Failed Failed Failed Failed 13 20180328ZY2569 Failed Failed Successful Successful 14 20180329MRL9449 Failed Failed Failed Successful 15 20180329LJX5553 Failed Failed Failed Successful 16 20180330WLJ3822 Failed Successful Successful Successful 17 20180402LRY7640 Failed Failed Failed Successful 18 20180402LY1226 Failed Failed Failed Successful 19 20180403LRZ5217 Failed Failed Successful Successful 20 20180404LGH0188 Failed Failed Successful Successful Summary 0% 20% 45% 80% (1/20) (4/20) (9/20) (16/20)

[0277] It can be seen that different sample dissociation solutions have a very great impact on the success rate for culturing primary cells in solid tumor tissues of gastric cancer, the sample dissociation solution used in the present invention (Table 3) can isolate the cancer cells in solid tumor tissues of gastric cancer to the greatest extent and improve the success rate for culturing primary cells in solid tumor tissues of gastric cancer.

Embodiment 15. Comparing Success Rates of Different Cell Digestion Solutions for Passaging Primary Cells of Gastric Cancer

[0278] The operation methods and processes for passaging primary cells of all samples in this embodiment were identical (as mentioned above), and the only difference was the cell digestion solution formula. Various tested sample dissociation solutions are shown in Table 34. Wherein Scheme D is the formula used in the present invention, and the details are shown in Table 7.

TABLE-US-00034 TABLE 34 Cell digestion solution formula for test (10 mL) Cell dissociation Final solution Reagent Brand Article No. Dosage Concentration Scheme A Trypsin Gibco# 25200056 10 mL Scheme B 0.5M EDTA Invitrogen#AM9261 10 μL 5 mM TrypLE ™ Express Gibco# 12604013 Replenish to 10 mL 10× collagenase I 10× stock solution 1 mL 200 U/mL 10× collagenase II 10× stock solution 1 mL 200 U/mL Scheme C 20× collagenase IV 20× stock solution 0.5 mL 100 U/mL PBS Gibco#21-040-CVR Replenish to 10 mL Scheme D See Table 7

[0279] The cell digestion solution is to be prepared just before use.

[0280] 20 successfully cultured samples of gastric cancer were selected, and the primary cells in solid tumor tissues of gastric cancer obtained by culture were continuously passaged with the above four cell digestion solutions respectively according to the method described in Embodiment 6. Passage (for no more than 10 times) was performed every time cancer cells amplified and formed cell masses with a diameter of 100 μm, and the maximum passage number was recorded. The statistical results are as shown in Table 35:

TABLE-US-00035 TABLE 35 Culturing in different cell digestion solutions Scheme Scheme Scheme Scheme S/N Sample No. A B C D 1 20180404DYZ6158 1 2 5 9 2 20180409XY0363 0 3 5 9 3 20180404LRJ6431 1 2 4 7 4 20180409LX57372 1 3 7 10 5 20180410LCZ4904 0 1 4 7 6 20180410WXX8514 1 4 7 10 7 20180411LYC9049 1 4 6 9 8 20180411Z5J2828 1 4 8 10 9 20180411GB9371 0 2 4 9 10 20180411ZFZ3083 1 3 6 10 11 20180413LJS1311 1 4 7 10 12 20180417XWY4460 1 3 8 10 13 20180419GFL5374 0 2 5 8 14 20180424ZLY5383 1 4 6 9 15 20180424YYC5496 0 1 5 7 16 20180425LSZ5296 1 4 6 9 17 20180426YCX7878 0 3 8 10 18 20180427LYQ2939 0 2 4 8 19 20180427GX6461 0 1 4 7 20 20180428CQF8086 1 4 7 10 Summary of 0-1 2-4 4-8 More than passage number time times times 7 times

[0281] It can be seen that different cell digestion solutions have a very great impact on the success rate for passaging primary cells in solid tumor tissues of gastric cancer, the cell digestion solution used in the present invention (Table 7) can mildly dissociate the cancer cells in the cell masses to continuously passage the samples and maintain the activity of primary cells in solid tumor tissues of gastric cancer.

Embodiment 16. Culturing Primary Tumor Cells of Gastric Cancer with Culture Consumables of Different Materials

[0282] The operation methods and processes of primary culture of all samples in this embodiment were identical (as mentioned above), and the only difference was the cell culture consumable material (unmodified) (Table 36).

TABLE-US-00036 TABLE 36 Effects of unmodified culture consumables of different materials on culturing primary tumor cells of gastric cancer S/N Sample No. PS PC PMMA COC COP LAS 1 20180428JLS0901 Successful Successful Successful Successful Failed Successful 2 20180428L5L4619 Failed Failed Failed Successful Failed Successful 3 20180503WZP4591 Successful Successful Successful Successful Successful Successful 4 20180504ZTL7916 Successful Failed Successful Successful Successful Successful 5 20189597NAY7068 Successful Successful Successful Successful Successful Successful 6 20180508CYM9714 Successful Failed Successful Failed Failed Successful 7 20180508ZQY8343 Failed Failed Failed Failed Failed Failed 8 20189510WSM9509 Successful Failed Successful Successful Successful Successful 9 20180511ZYJ2145 Successful Successful Successful Successful Successful Successful 10 20180511SYR5862 Failed Failed Failed Failed Failed Failed Successful 7/10 4/10 7/10 7/10 5/10 8/10 Note: Polystyrene (abbreviated as PS), Polycarbonate (abbreviated as PC), poly-methyl methacrylate (abbreviated as PMMA), COC resin, Cyclo Olefin Polymer (abbreviated as COP), low-attachment-surface (abbreviated as LAS).

[0283] It can be seen from Table 36 that culture consumables of different materials have a certain impact on the success rates for culturing primary cells in solid tumor tissues of gastric cancer, wherein the success rate of low-attachment-surface (LAS) is the highest.

Embodiment 17. Culturing Primary Tumor Cells of Gastric Cancer with CYTOP-Modified Culture Consumables

[0284] The operation methods and processes of primary culture of all samples in this embodiment were identical (as mentioned above), the CYTOP modification methods were identical, and the only difference was the culture consumable material (Table 37).

[0285] Wherein the CYTOP modification method was that: firstly, pure oxygen etching was performed on the cell culture vessel at a power of 20 W power for 3 minutes. Then the surface of a culture dish or culture plate was covered with an appropriate amount (taking a 96-well plate as an example, with 20 μL per well, an appropriate amount refers to complete coverage of the bottom of the culture dish) of 1% CYTOP solution, and the vessel could be used after the CYTOP solution was completely dried in the air.

TABLE-US-00037 TABLE 37 Effects of CYTOP-modified culture consumables of different materials on culturing primary tumor cells of gastric cancer S/N Sample No. PS PC PMMA COC COP LAS 1 20180517GZX3591 Successful Successful Successful Successful Successful Successful 2 20180517GQL6334 Successful Successful Successful Successful Successful Successful 3 20180518ZXF2420 Successful Successful Successful Successful Successful Successful 4 20180518EJF5851 Failed Failed Failed Failed Failed Failed 5 20180518WBY1609 Successful Successful Successful Successful Successful Successful 6 20180521LXL3551 Successful Successful Successful Successful Successful Successful 7 20180522SJD6275 Successful Successful Successful Successful Successful Successful 8 20180523GSH2064 Successful Successful Successful Successful Successful Successful 9 20180524ZCS2872 Successful Successful Successful Successful Successful Successful 10 20180524XGY3833 Successful Successful Successful Successful Successful Successful Success rate 9/10 9/10 9/10 9/10 9/10 9/10

[0286] It can be seen from Table 37 that CYTOP modification can effectively improve the success rates of various materials.

Example 18. Performing Drug Sensitivity Test with Primary Tumor Cells of Gastric Cancer

[0287] All the chemotherapeutic agents used in this embodiment, i.e. Irinotecan, 5-Fluorouracil and Oxaliplatin, are Selleck products.

[0288] The Celltiter-Glo cell viability test kit mentioned in this embodiment is a Promega product.

[0289] An in vitro drug sensitivity test was performed for the primary cells of gastric cancer cultured in the present invention: primary cells were inoculated at a density of 10.sup.5/well in a 96-well low-attachment cell culture plate of standard size, and 5 drug concentration gradients were set for each drug, n=3. After dosing, the cells were incubated under the condition of 37° C., 5% CO.sub.2 for 7 days. Following the end of drug action, the cell viability in each well was detected by the Celltiter-Glo cell viability assay kit. The experimental results are as shown in FIG. 6. The results indicate that the primary cells of gastric cancer obtained by the present method can be used for in vitro drug sensitivity test.

Embodiment 19. Preparing a Reagent for Culturing Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0290] 1. Sample preservation solution (100 mL)

[0291] For the specific formula (Table 1) and preparation method of sample preservation solution (100 mL), see step 1 in Embodiment 1.

[0292] 2. Sample washing solution (100 mL)

[0293] For the specific formula (Table 2) and preparation method of sample washing solution (100 mL), see step 2 in Embodiment 1.

[0294] 3. Sample dissociation solution (10 mL)

[0295] For the specific formula (Table 3) and preparation method of sample dissociation solution (10 mL), see step 3 in Embodiment 1.

[0296] 4. Cell digestion solution (10 mL)

[0297] For the specific formula (Table 7) and preparation method of cell digestion solution (10 mL), see step 4 in Embodiment 1.

[0298] 5. Digestion termination solution (100 mL)

[0299] For the specific formula (Table 8) and preparation method of digestion termination solution (100 mL), see step 5 in Embodiment 1.

[0300] 6. Primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma (100 mL)

[0301] For the specific formula (Table 9) and preparation method of primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma (100 mL), see step 6 in Embodiment 1.

[0302] 7. Cell cryopreserving solution

[0303] For the specific formula (Table 24) and preparation method of cell cryopreserving solution, see step 7 in Embodiment 1.

[0304] 8. 1% CYTOP solution

[0305] For the specific formula (Table 26) and preparation method of 1% CYTOP solution, see step 8 in Embodiment 1.

Embodiment 20. Obtaining Postoperative Specimens/Biopsy Puncture Specimens of Gallbladder Cancer and Cholangiocarcinoma

[0306] 1. The inventor cooperated with national triple A, first-class hospitals in China, and the cooperation passed formal medical ethical review.

[0307] 2. The attending physician selected the enrolled patients according to the clinical indications specified in the medical guidelines, and selected suitable samples for in vitro culture according to the intraoperative clinical indications. The selection criteria of samples during surgery were as follows: primary gallbladder cancer or cholangiocarcinoma, with pathological staging of stage II, III or IV, metastatic lesions of gallbladder cancer and cholangiocarcinoma of various pathological types, and samples with a surgical specimen weighing more than 20 mg. The selection criteria of biopsy puncture samples were as follows: primary gallbladder cancer or cholangiocarcinoma, with pathological staging of stage II, III or IV, metastatic lesions of gallbladder cancer and cholangiocarcinoma of various pathological types, and samples with more than 4 puncture specimens.

[0308] 3. The attending physician provided the patient's basic clinical information, such as gender, age, medical history, family history, smoking history, pathological stages and types and clinical diagnosis. The information related to patient privacy, such as patient's name and ID number, was concealed and replaced by a unified experimental number, and the naming principle of the experimental number was the 8-digit date of sample collection+last four digits of the patient's admission number. For example, for a sample provided on Jan. 1, 2018, when the patient's admission number was T001512765, then the sample experiment number was 201801012765.

[0309] 4. During the operation, a surgeon collected a fresh surgical specimen in a sterile environment of an operating room and placed it in a sample preservation solution prepared in advance (see Embodiment 19). After being detached, the sample was temporarily stored on ice and transported to a laboratory within two hours for the next operation.

[0310] 5. A puncture physician collected a fresh puncture specimen in a sterile environment of a puncture operating room and placed it in a sample preservation solution prepared in advance (see Embodiment 19). After being detached, the sample was temporarily stored on ice and transported to a laboratory within two hours for the next operation.

Embodiment 21. Performing Predissociation Treatment for Samples of Gallbladder Cancer and Cholangiocarcinoma

[0311] The following steps were operated on ice, and the whole operation process was completed within 10 minutes.

[0312] All the surgical apparatuses used in the following actions were used only after high-temperature and high-pressure sterilization and oven drying in advance.

[0313] 1. The sample was weighed.

[0314] 2. The sample surface was washed with 75% (volume percentage) ethanol for 10 to 30 seconds.

[0315] 3. The sample was washed with sample washing solution for 10 times and with sterile PBS solution for 5 times.

[0316] 4. Fatty tissues, connective tissues and necrotic tissues in the sample were carefully peeled off with ophthalmic scissors, ophthalmic forceps, scalpel and other apparatuses.

Embodiment 22. Dissociating Tissue Samples of Gallbladder Cancer and Cholangiocarcinoma

[0317] All the surgical apparatuses used in the following actions were used only after high-temperature and high-pressure sterilization and oven drying in advance.

[0318] 1. The tissues were cut into small pieces of about 1 mm.sup.3 with ophthalmic scissors.

[0319] 2. The tissue sample cut into pieces was treated with sample dissociation solution preheated to 37° C. in advance according to the dosage of 0.1 mL of sample dissociation solution (see Embodiment 19) per mg of tissue, and the sample was dissociated at 37° C. for 15 minutes to 3 hours. Observing the dissociation of the sample under a microscope every 15 minutes until a large number of individual cells are observed.

[0320] 3. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 19), and the cell suspension was collected.

[0321] 4. The cell suspension was filtered with a 40 μm sterile cell strainer to remove tissue fragments and adherent cells.

[0322] 5. The cell suspension was centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0323] 6. The cells were resuspended with 5 mL of sterile PBS and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0324] 7. The cell precipitation was resuspended with the primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma (see Embodiment 19), the cell state was observed under a microscope, and the cells were counted.

[0325] In addition to tumor cells, there were also a large number of various types of other cells, such as red blood cells, lymphocytes and fiber cells, mixed in the single cell suspension obtained by dissociation, as shown in FIG. 7. One of the advantages of the present method is that only the cancer cells could be amplified in the subsequent culture process, the proportion of other cells gradually decreases or even disappears, and primary tumor cells of gallbladder cancer and cholangiocarcinoma with a high purity are finally obtained.

Embodiment 23. Culturing Primary Cells of Gallbladder Cancer and Cholangiocarcinoma

[0326] 1. A low-attachment-surface was used for suspension-culturing primary cells in solid tumor tissues of gallbladder cancer and cholangiocarcinoma, and the culture medium in use was the primary cell culture medium for gallbladder cancer and cholangiocarcinoma in Embodiment 19 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein noggin was 100 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM); a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

[0327] 2. The cell state was observed every day, and the culture medium was replaced every 3 days until the cells formed masses with a diameter of about 80 μm.

[0328] As shown in FIG. 8, after 3-10 days of culture, cancer cells were amplified, forming cell masses with a diameter of 80 μm, the total number of tumor cells exceeded 10.sup.7, and the number of other types of cells was significantly reduced or even disappeared. After a large number of sample tests, the success rate of the present method for culturing in vitro primary tumor cells of gallbladder cancer and cholangiocarcinoma could reach 80%.

Embodiment 24. Passaging Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0329] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0330] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0331] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 19) and digested at 37° C. The digestion of cell masses was observed under the microscope every 5 minutes until all the cell masses were digested into individual cells.

[0332] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 19), and the cell suspension was collected.

[0333] 5. The cell suspension was centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0334] 6. The cell precipitation was resuspended with the primary cell culture medium of gallbladder cancer and cholangiocarcinoma, and the cells were counted.

[0335] 7. A low-attachment-surface was used for culturing primary cells of gallbladder cancer and cholangiocarcinoma, and the culture medium in use was the primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma in Embodiment 19; a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 25. Cryopreserving Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0336] The primary cells in solid tumor tissues of gallbladder cancer and cholangiocarcinoma suspension-cultured could be frozen after 2-3 passages and amplifications:

[0337] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0338] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0339] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 19) and digested at 37° C. The digestion of cell masses was observed under the microscope every 15 minutes until all the cell masses were digested into individual cells.

[0340] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 19), the cell suspension was collected, and the cells were counted.

[0341] 5. The cell suspension was centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0342] 6. The cell precipitation was resuspended at a density of 10.sup.6/mL with cell cryopreserving solution (see Embodiment 19), each 2 mL cryopreserving tube contained 1 mL of cell suspension, and the cell suspension was frozen overnight in a gradient cooling box and transferred into liquid nitrogen for long-term preservation.

Embodiment 26. Resuscitating Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0343] Primary cells in solid tumor tissues of gallbladder cancer and cholangiocarcinoma preserved in liquid nitrogen were resuscitated:

[0344] 1. 37° C. sterile water was prepared five minutes in advance.

[0345] 2. The cryopreserving tubes were removed from liquid nitrogen, and the cells were quickly melted in 37° C. sterile water.

[0346] 3. The cell solution was centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0347] 4. The cell precipitation was resuspended with the primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma (see Embodiment 19), the primary cells of gallbladder cancer and cholangiocarcinoma were cultured using a low-attachment-surface, the cells in each tube were resuscitated into a 3.5 cm culture dish, and the cells were cultured in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 27. HE Staining Identification of Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0348] Description of reagent consumables to be used in the following embodiment:

[0349] HE Staining Kit (Beijing Solarbio Science & Technology Co., Ltd., #G1120);

[0350] Cationic anticreep slide (ZSGB-BIO);

[0351] Xylene, methanol, acetone (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure);

[0352] Neutral resin adhesive (Beijing Yili Fine Chemicals Co., Ltd.).

[0353] 1. Primary cell masses from solid tumor tissues of gallbladder cancer and cholangiocarcinoma obtained by culturing with the primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma in Embodiment 19 (wherein the final concentration of human recombinant protein EGF was 20 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of human recombinant protein Noggin was 100 ng/mL; the final concentration of SB202190 was 5 μM; the final concentration of A83-01 was 1 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were collected by centrifugation at 800 g, and immobilized with 4% paraformaldehyde. Cell mass precipitation was imbedded in paraffin and sectioned with a thickness of 5 μm.

[0354] 2. Paraffin sections were immersed in dimethylbenzene solution and incubated at a room temperature for 5 minutes for dewaxing, and after this process was repeated for 3 times, the sections were flushed with deionized water twice.

[0355] 3. The sections were immersed in absolute ethanol and incubated at a room temperature for 10 minutes, and this process was repeated twice.

[0356] 4. The sections were immersed in 95% ethanol and incubated at a room temperature for 10 minutes, and after this process was repeated twice, the sections were flushed with deionized water twice.

[0357] 5. 100 μL of hematoxylin staining solution was added for staining for 1 min when the moisture on the slide was slightly dried.

[0358] 6. The hematoxylin staining solution was absorbed, and the slide was washed for 3 times with tap water.

[0359] 7. 100 μL of differentiation solution was added dropwise for differentiation for 1 min.

[0360] 8. The differentiation solution was absorbed, the slide was washed twice with tap water and washed with distilled water once.

[0361] 9. The moisture on the slide surface was absorbed, and 200 μL of eosin stain was added dropwise for staining for 40 s.

[0362] 10. The eosin stain was absorbed, and rinsing and dehydration were performed successively with 75%, 80%, 90% and 100% ethanol for 20 s, 20 s, 40 s and 40 s.

[0363] 11. After the ethanol was dried in the air, 50 μL of dimethylbenzene was added dropwise for cell permeability.

[0364] 12. After dimethylbenzene was completely dried, a drop of neutral resin adhesive was added, and the slide was sealed with a cover glass, observed under the microscope and photographed.

[0365] FIG. 9 shows an HE staining effect image of primary tumor cells of cholangiocarcinoma obtained by in vitro culture. It can be seen that these cells generally have the characteristics of cancer cells, such as high nuclear cytoplasmic ratio, hyperchromasia, chromatin condensation in nucleus, multinucleation and uneven cell size, and dozens to hundreds of tumor cells gather to form tumor cell masses with a certain three-dimensional structure.

Embodiment 28. Immunohistochemical Staining Identification of Primary Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma

[0366] Description of reagents to be used in the following embodiment:

[0367] Paraformaldehyde (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure), paraformaldehyde powder was dissolved with ultrapure water to form 4% (4 g/100 mL paraformaldehyde solution;

[0368] Hydrogen peroxide (Beijing Beihua Zhongtuo Technology Co., Ltd., 35%);

[0369] Normal goat serum for blocking (Solarbio, SL038);

[0370] Immunofluorescence primary antibody (Abcam, ab215838);

[0371] Immunofluorescence secondary antibody (Abcam, ab205719);

[0372] EDTA repair solution (Abcam, ab93684);

[0373] DAB chromogen (SignalStain® DAB Substrate Kit, 8059S)

[0374] The cell masses of gallbladder cancer and cholangiocarcinoma obtained by culturing with the primary cell culture medium for solid tumor tissues of gallbladder cancer and cholangiocarcinoma in Embodiment 19 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 25 ng/mL; the final concentration of human recombinant protein HGF was 25 ng/mL; the final concentration of human recombinant protein FGF-10 was 25 ng/mL; the final concentration of human recombinant protein Wnt-3a was 300 ng/mL; the final concentration of human recombinant protein Noggin was 200 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were collected for paraffin section, and epithelial-derived cells were characterized with pan-CK antibody according to the following steps.

[0375] 1. The section was successively immersed in dimethylbenzene I for 10 min and dimethylbenzene II (10 min).

[0376] 2. The section was immersed in absolute ethyl alcohol I (5 min)—absolute ethyl alcohol II (5 min)—95% alcohol (5 min)—80% alcohol (5 min)—70% alcohol (5 min), and then flushed with deionized water twice, 2 min each time.

[0377] 3. The tissue section was put into a repair box, then an appropriate amount of diluted EDTA repair solution (pH 9.0) was added, with the level higher than the tissue section.

[0378] 4. The tissue section was repaired with microwave mid-range for 10 min (time started when the liquid boiled), and a dry tissue should be avoided in this process.

[0379] 5. The repair box was taken out of the microwave oven and cooled down in air, and after the repair solution dropped to the room temperature, the glass slide was taken out and flushed with PBS (pH 7.4) for 3 times, 3 min each time (do not flush toward the tissue in the flushing process to avoid breaking the tissue).

[0380] 6. Prepared 3% hydrogen peroxide (30% hydrogen peroxide diluted with deionized water) was added to the section tissue dropwise to block endogenous peroxidase, incubated at a room temperature for 15 min, and flushed with PBS for 3 times, 3 min each time.

[0381] 7. PBS was dried out with absorbent paper, 10% goat serum (consistent with or similar to the species source of the secondary antibody) was added dropwise on the glass slide, and the glass slide was blocked at 37° C. for 60 min.

[0382] 8. The liquid around the tissue was wiped dry with absorbent paper, a circle was drawn around the tissue by mark pen, then the diluted primary antibody was added dropwise, and the slide was incubated in a wet box at 4° C. overnight.

[0383] 9. The section was flushed with PBS for 3 times, 3 min each time, a horseradish peroxidase labeled secondary antibody was added dropwise after the section was wiped dry with absorbent paper, and the section was incubated at a room temperature for 60 min.

[0384] 10. The section was flushed with PBS for 3 times, 3 min each time, the section was wiped dry with absorbent paper after PBS liquid was thrown backward, freshly-prepared DAB chromogen was added to each section dropwise, the sections were observed under a microscope, and the section was flushed with tap water after a positive signal was shown to stop the color development.

[0385] 11. Hematoxylin counterstaining was performed for 1 min, and the section was differentiated with acid ethanol differentiation solution after being washed with water, and flushed with tap water until cell nucleus turned blue.

[0386] 12. After being put into water and flushed, the section was successively put into: 70% alcohol—80% alcohol—90% alcohol—95% alcohol—absolute ethyl alcohol I—absolute ethyl alcohol II—dimethylbenzene I—dimethylbenzene II for dehydration and clearing, 2 min in each reagent, and finally the section was dried in a fume cupboard.

[0387] 13. The section was sealed with neutral balsam and covered with cover glass. The section was placed in a fume cupboard and dried.

[0388] 14. The dried section was observed or photographed under a microscope.

[0389] FIG. 10 shows an immunofluorescence staining effect image of primary tumor cell masses of cholangiocarcinoma cultured in vitro. It can be seen that all the cells constituting the cell masses are pan-CK positive and epithelial-derived, which confirms that the tumor cells obtained by the present method are those with a high purity Immunofluorescence staining identification was performed on 5 primary cultures of gallbladder cancer and cholangiocarcinoma samples, and the statistical results showed that the proportion of tumor cells in the primary cells of gallbladder cancer and cholangiocarcinoma obtained in the present method reached 84%-95% (Table 38).

TABLE-US-00038 Embodiment 38 Immunohistochemical staining identification of primary cultures of gallbladder cancer and cholangiocarcinoma Positive Rate of S/N Sample No. Type Pan-CK 1 20181127LXG2000 cholangiocarcinoma 95% 2 20190225LZL1104 Cholangiocarcinoma 84% 3 20190225SHM1105 Cholangiocarcinoma 89% 4 20190514JJK1147 Gallbladder cancer 91% 5 20190712HHL0332 Cholangiocarcinoma 87%

Embodiment 29. Culturing Primary Tumor Cells in Solid Tumor Tissues of Gallbladder Cancer and Cholangiocarcinoma with CYTOP-Modified Culture Consumables

[0390] The operation methods and processes of primary culture of all samples in this embodiment were identical (as mentioned above), the CYTOP modification methods were identical, and the only difference was the cell culture consumable material (Table 39).

[0391] Wherein the CYTOP modification method was that: firstly, pure oxygen etching was performed on the cell culture vessel at a power of 20 W for 3 minutes. Then the surface of a culture dish or culture plate was covered with an appropriate amount (taking a 96-well plate as an example, with 20 μL per well, an appropriate amount refers to complete coverage of the bottom of the culture dish) of 1% CYTOP solution, and the vessel could be used after the CYTOP solution was completely dried in the air.

TABLE-US-00039 TABLE 39 Effects of CYTOP-modified consumables on culturing primary tumor cells in solid tumor tissues of gallbladder cancer and cholangiocarcinoma PS CYTOP S/N Sample No. Type PS modification 1 20190321CZ1114 Cholangiocarcinoma Failed Successful 2 20190823WJW0401 Cholangiocarcinoma Failed Successful 3 20191009LCH0776 Gallbladder cancer Failed Successful 4 20190923H5E0482 Gallbladder cancer Failed Failed 10 20190924WMY0335 Cholangiocarcinoma Failed Successful Success rate 0/5 4/5 Note: Polystyrene (abbreviated as PS).

[0392] It can be seen from Table 39 that CYTOP modification can improve the success rate for culturing samples greatly.

Embodiment 30. Preparing a Reagent for Culturing Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0393] 1. Cell Isolation Buffer (100 mL)

[0394] The specific formula of cell isolation buffer (100 mL) is shown in Table 40:

TABLE-US-00040 TABLE 40 Cell isolation buffer (100 mL) Final Reagent Brand Article No. Dosage Concentration P/S Gibco#15140122 1 mL 1% Heparin sodium 1000× stock solution 1 mL 10 IU/mL solution PBS Gibco#21-040-CVR Replenish to 100 mL

[0395] The prepared cell isolation buffer can be preserved at 4° C. for 1 month.

[0396] In Table 40, the preparation of heparin sodium solution is shown in Table 41.

TABLE-US-00041 TABLE 41 1000× heparin sodium (1 mL) Final Reagent Brand Article No. Dosage Concentration Heparin sodium Solarbio#H8270 10 kIU 10 IU/L Ultrapure water Replenish to 1 mL 1000× heparin sodium solution is to be prepared just before use.

[0397] 2. Cell Digestion Solution (10 mL)

[0398] For the specific formula (Table 7) and preparation method of cell digestion solution (10 mL), see step 4 in Embodiment 1.

[0399] 3. Digestion Termination Solution (100 mL)

[0400] For the specific formula (Table 8) and preparation method of digestion termination solution (100 mL), see step 5 in Embodiment 1.

[0401] 4. Primary Cell Culture Medium for Bile Sample of Gallbladder Cancer and Cholangiocarcinoma (100 mL)

[0402] For the specific formula (Table 9) and preparation method of primary cell culture medium for the bile sample of gallbladder cancer and cholangiocarcinoma (100 mL), see step 6 in Embodiment 1.

[0403] 5. Cell Cryopreserving Solution

[0404] For the specific formula (Table 24) and preparation method of cell cryopreserving solution, see step 7 in Embodiment 1.

Embodiment 31. Obtaining Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0405] 1. The inventor cooperated with national triple A, first-class hospitals in China, and the cooperation passed formal medical ethical review.

[0406] 2. The attending physician selected the enrolled patients according to the clinical indications specified in the medical guidelines, and selected suitable samples for in vitro culture according to the intraoperative clinical indications. The selection criteria of samples during surgery were as follows: sample of primary gallbladder cancer or cholangiocarcinoma, with pathological staging of stage II, III or IV, and bile sample volume of more than 20 mL.

[0407] 3. The attending physician provided the patient's basic clinical information, such as gender, age, medical history, family history, smoking history, pathological stages and types and clinical diagnosis. The information related to patient privacy, such as patient's name and ID number, was concealed and replaced by a unified experimental number, and the naming principle of the experimental number was the 8-digit date of sample collection+last four digits of the patient's admission number. For example, for a sample provided on Jan. 1, 2018, when the patient's admission number was T001512765, then the sample experiment number was 201801012765.

[0408] 4. The doctor in charge of the patient collected more than 10 mL of fresh bile sample with sterile apparatus. The sample was temporarily stored on ice and transported to a laboratory for the next operation within 48 hours.

Embodiment 32. Pretreating Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0409] The following steps were operated on ice, and the whole operation process was completed within 10 minutes.

[0410] 1. The bile sample was kept still on ice for about 30 minutes, so that blood clots and large insoluble solids in the sample were settled to the bottom of the sample tube;

[0411] 2. The supernatant was carefully transferred into a 50 mL sterile centrifuge tube, and a 1× volume of precooled PBS was added and blended;

[0412] 3. The cell suspension was centrifuged at 2,000 g at 4° C. for 5 minutes, and the supernatant was discarded;

[0413] 4. The cell precipitation was resuspended with cell separation buffer (see Embodiment 30) and centrifuged at 2,000 g at 4° C. for 5 minutes, and the supernatant was discarded;

[0414] 5. The cell precipitation was resuspended with cell separation buffer (see Embodiment 30), and the cell concentration was adjusted to 10.sup.7/mL.

Embodiment 33. Density Gradient Centrifugation of Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0415] 1. Ficoll cell separation solution (MP #50494) of the same volume as the cell suspension was taken by a 50 mL sterile centrifuge tube.

[0416] 2. The cell suspension was carefully added to the upper layer of the cell separation solution, so that a clear interface was formed between them.

[0417] 3. The cell suspension was horizontally centrifuged at 2,000 at a room temperature for 20 minutes.

[0418] 4. A white membrane in an intermediate layer was sucked into a new tube.

[0419] 5. The cell precipitation was resuspended with 20 mL sterile PBS and centrifuged at 1,500 g at a room temperature for 10 minutes, and the supernatant was discarded.

[0420] 6. The cell precipitation was resuspended with the primary cell culture medium for the bile samples of gallbladder cancer and cholangiocarcinoma (see Embodiment 30), the cell state was observed under a microscope, and the cells were counted.

[0421] In addition to tumor cells, there were also a large number of various types of other cells, such as red blood cells, lymphocytes and fiber cells, mixed in the single cell suspension obtained by separation, as shown in FIG. 11. One of the advantages of the present method is that only the cancer cells could be amplified in the subsequent culture process, the proportion of other cells gradually decreases or even disappears, and primary tumor cells of gallbladder cancer and cholangiocarcinoma with a high purity are finally obtained.

Embodiment 34. Culturing Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0422] 1. A low-attachment-surface was used for suspension-culturing primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma, and the culture medium in use was the primary cell culture medium for the bile samples of gallbladder cancer and cholangiocarcinoma in Embodiment 30 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 250 ng/mL; the final concentration of human recombinant protein noggin was 100 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM); a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

[0423] 2. The cell state was observed every day, and the culture medium was replaced every 3 days until the cells formed masses with a diameter of about 80 μm.

[0424] As shown in FIG. 12, after 3-10 days of culture, cancer cells were amplified, forming cell masses with a diameter of 80 μm, the total number of tumor cells exceeded 10.sup.7, and the number of other types of cells was significantly reduced or even disappeared. After a large number of sample tests, the success rate of the present method for culturing in vitro primary tumor cells in the bile sample of gallbladder cancer and cholangiocarcinoma could reach 70%.

Embodiment 35. Passaging Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0425] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0426] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0427] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 30) and digested at 37° C. The digestion of cell masses was observed under the microscope every 5 minutes until all the cell masses were digested into individual cells.

[0428] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 30), and the cell suspension was collected.

[0429] 5. The cell suspension was centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0430] 6. The cell precipitation was resuspended with the primary cell culture medium of gallbladder cancer and cholangiocarcinoma, and the cells were counted.

[0431] 7. A low-attachment-surface was used for culturing primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma, and the culture medium in use was the primary cell culture medium for bile samples of gallbladder cancer and cholangiocarcinoma in Embodiment 30; a 6-well plate was taken as an example, and cells were planked at the density of 10.sup.6 cells per well in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 36. Cryopreserving Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0432] The primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma suspension-cultured could be frozen after 2-3 passages and amplifications:

[0433] 1. The cell masses in a culture dish were collected and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0434] 2. The cell masses were washed with sterile PBS solution and centrifugated at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0435] 3. The cell masses were resuspended with cell digestion solution (see Embodiment 30) and digested at 37° C. The digestion of cell masses was observed under the microscope every 15 minutes until all the cell masses were digested into individual cells.

[0436] 4. The dissociation reaction was terminated with a 10× volume of digestion termination solution (see Embodiment 30), the cell suspension was collected, and the cells were counted.

[0437] 5. The cell suspension were centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0438] 6. The cell precipitation was resuspended at a density of 10.sup.6/mL with cell cryopreserving solution (see Embodiment 30), each 2 mL cryopreserving tube contained 1 mL of cell suspension, and the cell suspension was frozen overnight in a gradient cooling box and transferred into liquid nitrogen for long-term preservation.

Embodiment 37. Resuscitating Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0439] Primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma preserved in liquid nitrogen were resuscitated:

[0440] 1. 37° C. sterile water was prepared five minutes in advance.

[0441] 2. The cryopreserving tubes were removed from liquid nitrogen, and the cells were quickly melted in 37° C. sterile water.

[0442] 3. The cell solution were centrifuged at 800 g at a room temperature for 10 minutes, and a supernatant was discarded.

[0443] 4. The cell precipitation was resuspended with the primary cell culture medium for the bile sample of gallbladder cancer and cholangiocarcinoma (see Embodiment 30), the primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma were cultured using a low-attachment-surface, the cells in each tube were resuscitated into a 3.5 cm culture dish, and the cells were cultured in a cell incubator under the condition of 37° C., 5% CO.sub.2.

Embodiment 38. HE Staining Identification of Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0444] Description of reagent consumables to be used in the following embodiment:

[0445] HE Staining Kit (Beijing Solarbio Science & Technology Co., Ltd., #G1120);

[0446] Cationic anticreep slide (ZSGB-BIO);

[0447] Xylene, methanol, acetone (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure);

[0448] Neutral resin adhesive (Beijing Yili Fine Chemicals Co., Ltd.).

[0449] 1. Primary cell masses from solid tumor tissues of gallbladder cancer and cholangiocarcinoma obtained by culturing with the primary cell culture medium for bile samples of gallbladder cancer and cholangiocarcinoma in Embodiment 30 (wherein the final concentration of human recombinant protein EGF was 20 ng/mL; the final concentration of human recombinant protein bFGF was 20 ng/mL; the final concentration of human recombinant protein HGF was 20 ng/mL; the final concentration of human recombinant protein FGF-10 was 20 ng/mL; the final concentration of human recombinant protein Wnt-3a was 200 ng/mL; the final concentration of human recombinant protein Noggin was 100 ng/mL; the final concentration of SB202190 was 5 μM; the final concentration of A83-01 was 1 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were collected by centrifugation at 800 g, and immobilized with 4% paraformaldehyde. Cell mass precipitation was imbedded in paraffin and sectioned with the thickness of 5 μm.

[0450] 2. Paraffin sections were immersed in dimethylbenzene solution and incubated at a room temperature for 5 minutes for dewaxing, and after this process was repeated for 3 times, the sections were flushed with deionized water twice.

[0451] 3. The sections were immersed in absolute ethanol and incubated at a room temperature for 10 minutes, and this process was repeated twice.

[0452] 4. The sections were immersed in 95% ethanol and incubated at a room temperature for 10 minutes, and after this process was repeated twice, the sections were flushed with deionized water twice.

[0453] 5. 100 μL of hematoxylin staining solution was added for staining for 1 min when the moisture on the slide was slightly dried.

[0454] 6. The hematoxylin staining solution was absorbed, and the slide was washed for 3 times with tap water.

[0455] 7. 100 μL of differentiation solution was added dropwise for differentiation for 1 min.

[0456] 8. The differentiation solution was absorbed, the slide was washed twice with tap water and washed with distilled water once.

[0457] 9. The moisture on the slide surface was absorbed, and 200 μL of eosin stain was added dropwise for staining for 40 s.

[0458] 10. The eosin stain was absorbed, and rinsing and dehydration were performed successively with 75%, 80%, 90% and 100% ethanol for 20 s, 20 s, 40 s and 40 s.

[0459] 11. After the ethanol was dried in the air, 50 μL of dimethylbenzene was added dropwise for cell permeability.

[0460] 12. After dimethylbenzene was completely dried, a drop of neutral resin adhesive was added, and the slide was sealed with a cover glass, observed under the microscope and photographed.

[0461] FIG. 13 shows an HE staining effect image of primary tumor cells in the bile sample of cholangiocarcinoma obtained by in vitro culture. It can be seen that these cells generally have the characteristics of cancer cells, such as high nuclear cytoplasmic ratio, hyperchromasia, chromatin condensation in nucleus, multinucleation and uneven cell size, and dozens to hundreds of tumor cells gather to form tumor cell masses with a certain three-dimensional structure.

Embodiment 39. Immunohistochemical Staining Identification of Primary Cells in the Bile Samples of Gallbladder Cancer and Cholangiocarcinoma

[0462] Description of reagents to be used in the following embodiment:

[0463] Paraformaldehyde (Beijing Beihua Zhongtuo Technology Co., Ltd., analytical pure), paraformaldehyde powder was dissolved with ultrapure water to form 4% (4 g/100 mL paraformaldehyde solution;

[0464] Hydrogen peroxide (Beijing Beihua Zhongtuo Technology Co., Ltd., 35%);

[0465] Normal goat serum for blocking (Solarbio, SL038);

[0466] Immunofluorescence primary antibody (Abcam, ab215838);

[0467] Immunofluorescence secondary antibody (Abcam, ab205719);

[0468] EDTA repair solution (Abcam, ab93684);

[0469] DAB chromogen (SignalStain® DAB Substrate Kit, 8059S)

[0470] The primary cell masses from the bile samples of gallbladder cancer and cholangiocarcinoma obtained by culturing with the primary cell culture medium for bile sample of gallbladder cancer and cholangiocarcinoma in Embodiment 30 (wherein the final concentration of human recombinant protein EGF was 50 ng/mL; the final concentration of human recombinant protein bFGF was 25 ng/mL; the final concentration of human recombinant protein HGF was 25 ng/mL; the final concentration of human recombinant protein FGF-10 was 25 ng/mL; the final concentration of human recombinant protein Wnt-3a was 300 ng/mL; the final concentration of human recombinant protein Noggin was 200 ng/mL; the final concentration of SB202190 was 10 μM; the final concentration of A83-01 was 0.5 μM; the final concentration of N-acetyl-L-cysteine was 1 mM; the final concentration of Nicotinamide was 10 mM; the final concentration of cortisol was 25 ng/mL; the final concentration of Y-27632 was 10 μM) were collected for paraffin section, and epithelial-derived cells were characterized with pan-CK antibody according to the following steps.

[0471] 1. The section was successively immersed in dimethylbenzene I for 10 min and dimethylbenzene II (10 min).

[0472] 2. The section was immersed in absolute ethyl alcohol I (5 min)—absolute ethyl alcohol II (5 min)—95% alcohol (5 min)—80% alcohol (5 min)—70% alcohol (5 min), and then flushed with deionized water twice, 2 min each time.

[0473] 3. The tissue section was put into a repair box, then an appropriate amount of diluted EDTA repair solution (pH 9.0) was added, with the level higher than the tissue section.

[0474] 4. The tissue section was repaired with microwave mid-range for 10 min (time started when the liquid boiled), and a dry tissue should be avoided in this process.

[0475] 5. The repair box was taken out of the microwave oven and cooled down in air, and after the repair solution dropped to the room temperature, the glass slide was taken out and flushed with PBS (pH 7.4) for 3 times, 3 min each time (do not flush toward the tissue in the flushing process to avoid breaking the tissue).

[0476] 6. Prepared 3% hydrogen peroxide (30% hydrogen peroxide diluted with deionized water) was added to the section tissue dropwise to block endogenous peroxidase, incubated at a room temperature for 15 min, and flushed with PBS for 3 times, 3 min each time.

[0477] 7. PBS was dried out with absorbent paper, 10% goat serum (consistent with or similar to the species source of the secondary antibody) was added dropwise on the glass slide, and the glass slide was blocked at 37° C. for 60 min.

[0478] 8. The liquid around the tissue slide was wiped dry with absorbent paper, a circle was drawn around the tissue by mark pen, then the diluted primary antibody was added dropwise, and the slide was incubated in a wet box at 4° C. overnight.

[0479] 9. The section was flushed with PBS for 3 times for 3 min each time, a horseradish peroxidase labeled secondary antibody was added dropwise after the section was wiped dry with absorbent paper, and the section was incubated at a room temperature for 60 min.

[0480] 10. The section was flushed with PBS for 3 times for 3 min each time, the section was wiped dry with absorbent paper after PBS liquid was thrown backward, freshly-prepared DAB chromogen was added to each section dropwise, the sections were observed under the microscope, and the section was flushed with tap water after a positive signal was shown to stop the color development.

[0481] 11. Hematoxylin counterstaining was performed for 1 min, and the section was differentiated with acid ethanol differentiation solution after being washed with water, and flushed with tap water until cell nucleus turned blue.

[0482] 12. After being put into water and flushed, the section was successively put into: 70% alcohol—80% alcohol—90% alcohol—95% alcohol—absolute ethyl alcohol I—absolute ethyl alcohol II—dimethylbenzene I—dimethylbenzene II for dehydration and clearing, 2 min in each reagent, and finally the section was dried in a fume cupboard.

[0483] 13. The section was sealed with neutral balsam and covered with cover glass. The section was placed in a fume cupboard and dried.

[0484] 14. The dried section was observed or photographed under a microscope.

[0485] FIG. 14 shows an immunofluorescence staining effect drawing of primary tumor cell masses of cholangiocarcinoma cultured in vitro. It can be seen that all the cells constituting the cell masses are pan-CK positive and epithelial-derived, which confirms that the tumor cells obtained by culture in the present method are those with a high purity. Immunofluorescence staining identification was performed on 5 primary cultures of gallbladder cancer and cholangiocarcinoma samples, and the statistical results showed that the proportion of tumor cells in the primary cells in the bile sample of gallbladder cancer and cholangiocarcinoma obtained in the present method reached 64%-80% (Table 42).

TABLE-US-00042 Embodiment 42 Immunohistochemical staining identification of primary cultures from the bile samples of gallbladder cancer and cholangiocarcinoma Positive Rate of S/N Sample No. Type Pan-CK 1 20190913LHY0460 Gallbladder cancer 77% 2 20190913WGH0461 Cholangiocarcinoma 64% 3 20190902YHQ0423 Cholangiocarcinoma 80% 4 20190805NZZ0126 Gallbladder cancer 69% 5 20190719WR1473 Cholangiocarcinoma 72%

Embodiment 40. Processing a Microplate Chip

[0486] In the embodiment, a microplate chip used for culturing the primary cells of gastric cancer, gallbladder cancer and cholangiocarcinoma in the present invention was made from PMMA material (or PS, PC, COC, COP, LAS and other materials) by injection molding. The chip can be used for culturing primary cells of gastric cancer, gallbladder cancer and cholangiocarcinoma and conducting in vitro drug sensitivity test. The design drawing of the microplate chip is as shown in FIG. 15.

[0487] Specifically, in the process of practical application, the microplate chip structure (design drawing as shown in FIG. 15) was prepared from PMMA material (or PS, PC, COC, COP, LAS and other materials), then the surface of the microplate chip was CYTOP-modified by the above CYTOP modification method (see Embodiment 29), so that the microplate chip available for culturing the primary cells of gastric cancer, gallbladder cancer and cholangiocarcinoma as stated herein was obtained.

INDUSTRIAL APPLICATION

[0488] The present invention provides a method for extracting and culturing primary tumor cells of gastric cancer, gallbladder cancer and cholangiocarcinoma from a fresh surgical sample of gastric cancer, a surgical sample or a biopsy puncture tissue sample of gallbladder cancer and cholangiocarcinoma, or a bile sample of gallbladder cancer and cholangiocarcinoma, and auxiliary reagents. The method has the following advantages: the tissue sample size is less, and only about 20 mg of surgical sample or about 10-20 mL of bile sample is required; the method can be used for either culturing primary tumor cells in primary tumors of gastric cancer, gallbladder cancer and cholangiocarcinoma, or culturing primary tumor cells in metastatic lesions of gastric cancer, gallbladder cancer and cholangiocarcinoma; the culture cycle is short, and the primary tumor cells with the order of magnitude of 10.sup.6-10.sup.7 can be obtained in only 3-10 days; the culture stability is high, and the success rate of the method for culturing in vitro qualified samples is as high as 70%; the cell purity is high, the proportion of cancer cells in the primary cell cultures of gastric cancer, gallbladder cancer and cholangiocarcinoma obtained by the method can be up to 60%-95%, with less interference from impurity cells. The primary cell cultures of gastric cancer, gallbladder cancer and cholangiocarcinoma obtained by the method of the present invention can be used in in vitro tests, next generation sequencing, construction of animal models, construction of cell lines and the like at various cell levels. It is predictable that such culture method will have a wide application prospect in the fields of research, clinical diagnosis and treatment of gastric cancer, gallbladder cancer and cholangiocarcinoma.